A technique has been developed in which a chimeric animal is produced from a hybrid cell obtained by fusion between a microcell containing a chromosome fragment and a pluriopotent cell (WO 97/07671). This enabled the production of a non-human animal carrying a very long foreign gene, which was heretofore impossible.
Modification of a chromosome fragment to be introduced into a non-human animal is useful because it realizes (1) removal of unnecessary genes, (2) addition of desired genes, (3) stabilization of a chromosome fragment and the like. WO 98/37757 describes a summary of a method for modifying a chromosome fragment to be introduced into a non-human animal and that a deletion chromosome of interest was obtained with high efficiency by targeting a telomeric sequence to a human chromosome retained in the DT-40 cell derived from a chicken. This publication also describes a fragment of a human chromosome which is stably retained in a mouse ES cell and an individual mouse, and has high genetic transmission efficiency. WO 00/10383 describes a method for producing a more stable human artificial chromosome (hereinafter this may be abbreviated to “HAC”) in which a desired region on the human chromosome is translocated to a stable chromosome fragment (chromosome vector).
Recently, Kuroiwa et al. (Nature Biotech. 18: 1086, 2000) succeeded, for the first time in the world, in producing a human artificial chromosome (HAC) retaining a specific human chromosome region of mega base (Mb) size as an insert. This HAC (λHAC) is an artificial chromosome that was obtained by using a SC20 fragment derived from human chromosome 14, which was stable and genetically transmissible, as a chromosome vector, and by translocating and cloning a 10 Mb chromosome region containing a human antibody λ light chain gene on human chromosome 22 to the vector as an insert. They demonstrated that this λHAC had a stability substantially equivalent to that of the SC20 fragment used as a vector and regions derived from various unstable chromosomes could be stabilized by being translocated and cloned to SC20 as well. Further, they introduced this λHAC to a mouse, thereby succeeding in producing a chimeric mouse which stably carried λHAC.
In a non-human animal, genetic transmission of an introduced human chromosome to the next generation is important not only with regard to mass-production of transchromosomic animals by crossing (i.e., a non-human animal in which heterogenic chromosome fragments have been genetically transmitted through a germ line) having homogeneity, but also with regard to analysis of structures and functions through a germ line of the introduced human chromosome. Several types of human chromosomes have been heretofore introduced into mice and the genetic transmission capacity thereof is considered to depend on the structure of the introduced human chromosome. For the purpose of genetic transmission, at the outset it is essential to obtain a chimeric mouse in which the ES cell contributes with high efficiency to a germ cell and the chimerism is high. This chimerism is considered to be associated with a structure of the introduced human chromosome, that is, which type of human gene is present on the introduced chromosome. For example, when a fragment of human chromosome 2 or 14 is introduced, a chimeric mouse whose chimerism is close to 100% is obtained and its genetic transmission efficiency is high (Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97: 722-727, 2000). In contrast, when a fragment of human chromosome 22 is introduced, a chimeric mouse whose chimerism is low, i.e., 50% or below, is obtained in most cases. This may be because a harmful human gene that adversely affects the development of a mouse is present on human chromosome 22. In fact, it is reported that gene expression-level-dependent hereditary disease-causing regions such as cat's eye syndrome, DiGeorge syndrome, and der22 syndrome exist in the 22q11 region on human chromosome 22 where the human antibody Ig λ gene is present (for example, A. Puech et al., PNAS 97: 10090, 2000). As described above, these hereditary disease-causing regions are removed, and only 10 Mb from the HCF2 locus to the LIF locus on human chromosome 22 is translocated and cloned to the SC20 chromosome vector to construct λHAC, followed by introduction into a mouse. As a result, the chimerism of the chimeric mouse generated from the ES cell retaining λHAC is reported to be enhanced compared to the case where the full length of human chromosome 22 was introduced.
Under the above circumstances, the present inventors have attempted to further improve the human artificial chromosome in order to achieve more efficient genetic transmission than the conventional λHAC, and have studied the genetic transmission efficiency.
More specifically, an object of the present invention is to provide a human artificial chromosome which is genetically transmissible to the next generation with high efficiency by modification of human chromosome 22 or a fragment thereof, and a non-human animal carrying the human artificial chromosome and an offspring thereof.
Another object of the present invention is to provide a method for producing a human antibody using the non-human animal or an offspring thereof.
The present inventors have conducted concentrated studies in order to attain the above objects. As a result, they have modified human chromosome 22, selected two types of regions with a clear construction containing an antibody λ light chain gene (Ig λ) region, and constructed a human artificial chromosome in which each of the selected regions was translocated to a fragment of human chromosome 14, thereby producing a mouse with a high chimerism carrying the same. As a result, the present inventors observed that the human artificial chromosome was genetically transmitted to the offspring at the next generation with high efficiency through meiosis in the chimeric mouse, thereby completing the present invention.